Abstract 2299: Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability
Authors: Haeyoung Kim, Mizuki Azuma
Journal: Cancer Research (2022)

Abstract Ewing sarcoma is the second most common bone cancer that develops in adolescents and young adults. Majority of Ewing sarcoma patients (~85%) carry an EWSR1-FLI1 fusion gene derived from t(11;22)(q24;q12) chromosomal translocation. The formation of EWSR1-FLI1 leads to the loss of one wild type EWSR1 allele, however, how loss of the gene contributes to the pathogenesis of Ewing sarcoma remains unclear. Our previous study revealed that the EWSR1 is required for a faithful mitosis. We demonstrated that the mutant ewsa (a homologue of human EWSR1) zebrafish induces mitotic defects, aneuploidy, and promotes tumorigenesis (with tp53 mutation). Consistently, Ewing sarcoma patients display high incidence of aneuploidy (e.g. approximately 47% of the patients display trisomy 8). For these reasons, we aim to elucidate the molecular function of EWSR1 in faithful chromosome segregation and in maintenance of chromosome stability. To accomplish our goal, we employed the Auxin-Inducible Degron (AID) system because it allows us to conditionally degrade EWSR1 proteins in a cell line. Both EWSR1 alleles were tagged with mini-AID using CRISPR/Cas9 system in a stable DLD-1 cell that expresses a plant E3 ligase TIR1. The western blot verified the efficient degradation of EWSR1 when the cells were treated with Auxin (AUX+) for 24hrs. To determine whether EWSR1 maintains chromosomal stability, the cells were synchronized to pro-metaphase using thymidine/nocodazole protocol, and were treated with AUX for 24hrs (underwent one cell cycle) concurrently. The EWSR1 knockdown (AUX+) cells displayed significantly higher incidence of lagging chromosomes compared to the control (AUX-) cells. Furthermore, immunocytochemistry using the antibody against the key mitotic regulator, Aurora B, revealed that the EWSR1 knockdown (AUX+) cells displayed significantly less incidence of Aurora B localization at inner centromere, but higher incidence of localization on kinetochore compared to the control (AUX-) cells. Consistently, when the cells that were treated with AUX for 48hrs (underwent two cell cycles), it displayed significantly higher incidence of aberrant numbers of chromosome compared to the control (AUX-) cells. In general, impaired chromosome segregation leads to mitotic delay, accompanied by the activation of mitotic checkpoint. However, the EWSR1 knockdown (AUX+) cells did not undergo any arrest in mitosis. Therefore, our data suggest that the EWSR1 knockdown (AUX+) cells not only induces aneuploidy by inducing lagging chromosome, but also it may override mitotic checkpoint. Currently, we are in a process of elucidating the molecular mechanism of EWSR1 knockdown dependent mitigation of mitotic arrest. Successful completion of the study may shed light on how/whether haploinsufficiency of EWSR1 contributes to the molecular pathogenesis of Ewing sarcoma. Citation Format: Haeyoung Kim, Mizuki Azuma. Function of Ewing sarcoma EWSR1 protein in the maintenance of chromosome stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2299.